The intracellular processing (cleavage and/or functional group modification) of precursor forms of native proteins following their translation from nucleic acid coding sequences has been clearly documented.
In general, mammalian cells and other eukaryotes can perform certain post-translational processing procedures, while prokaryotes can not. Certain prokaryotes, such as E. coli, are widely employed as hosts for the production of mammalian proteins via recombinant DNA (rDNA) technology because they can be readily grown in batch fermentation procedures and because they are genetically well-characterized. However, many mammalian proteins produced by genetic engineering technoloy require some type of post-translational processing, and this must often be accomplished by using complex, in vitro chemical or enzymatic procedures which are cost-prohibitive for large-scale production applications.
One type of processing activity involves the specific amidation (conversion of --COOH group to a --CONH.sub.2 group) of the carboxyl-terminal amino acid of a protein. Many naturally-occurring hormones and peptides contain such a modification, which is often essential if the protein is to be biologically active. An example is calcitonin, where the substitution of a non-amidated proline residue for the amidated proline of the native form results in a 3,000-fold reduction in biological activity.
The agent which effects this C-terminal (alpha) amidation recognizes a glycine residue which immediately follows the amino acid to be amidated (R-X-gly, where R is the main body of the protein, X is the residue which is amidated, and "gly" is the glycine residue). The glycine is cleaved and actually donates the amino moiety to the penultimate amino acid, thereby amidating it.
Enzymatic preparations capable of amidating the carboxyl-terminus of peptides and proteins have been described from a variety of sources. For instance. Bradbury, A. F., et al, Nature 298, 1982, p. 686-688 report an .alpha.-amidating enzyme activity to be present in porcine pituitary.
Husain, I., and Tate, S. S., FEBS Letters, Vol. 152, #2, 1983, p. 277-281, described an .alpha.-amidating activity present in bovine pituitary neurosecretory granules.
Eipper et al, PNAS Vol. 80, 1983, p. 5144-5148, reported an .alpha.-amidating enzyme activity to be present in the anterior, intermediate and posterior lobes of the rat pituitary gland.
Gomez et al, FEBS Letters, Vol. 167, #1, 1984, p. 160-164 determined that rat hypothalamus also contained an .alpha.-amidating enzyme activity.
Bradbury, A. F., Smythe, D. G., in Peptides Structure and Function: Proceedings of the Eighth American Peptide Symposium; p. 249-52 (1983), Editors Hruby, V. J., and Rich, D. H., describe the presence of an .alpha.-amidating enzyme activity in rat thyroid glands.
Mains R. E. et al, Endocrinology, Vol. 114, 1984, p. 1522-1530, reported that a murine cell line derived from the anterior pituitary lobe (ATT-20) contained an .alpha.-amidating enzyme activity that apparently decreased with time in culture.
Glands or organs known to contain amidated peptides may contain an enzyme capable of catalyzing the amidation reaction. For example, lower life forms such as the dog fish (Squalus acanthias) have been reported by O'Donohue T. L., et al, Peptides 3, 1982, p. 353-395, to contain amidated peptides in pituitary extracts. Scheller, R. H. et al, Cell, Vol. 32, 1983, p. 7-22 reported the presence of amidation signal peptides in the marine snail Apylsia. Despite the apparent ubiquitous distribution of this activity in nature, little information has been published on its physicochemical characteristics. This may be attributed to the very low levels of enzyme present in these neuroendocrine organs.
Heretofore, the purification and characterization of the .alpha.-amidating enzyme have not been published. Physicochemical properties of partially purified enzyme preparations, however, have been reported.
The first authors to report an approximate molecular weight for the .alpha.-amidating enzyme were Bradbury A. F., et al, Nature, Vol. 298, 1982, p. 686-88. Using Sephadex G-100 they suggested a minimum apparent molecular mass of approximately 60,000 daltons.
Subsequent studies have suggested the molecular mass of the enzyme to be between 60,000 and 70,000 daltons. These include Husain, I., and Tate S. S., FEBS Letters, Vol. 152. #2, 1983, p. 277-281; Eipper B. A., PNAS Vol. (16), 1983 p. 5144-5148; Gomez et al., FEBS Letters , Vol., 167, #1, 1984, p. 160-64, and Kizer J. S., et al, PNAS, Vol. 81, 1984, p. 3228-3232.
Eipper et al, PNAS, Vol. 80, 1983, p. 5144-48, have reported that in addition to molecular oxygen, two cofactors are required for maximal enzyme activity; these are ascorbic acid and copper (II) ion.
The chemical reaction resulting in the amidation of the carboxyl-terminus of a peptide requires a source for the amino group. Bradbury, A. F., et al, Nature, Vol. 298, 1982, p. 686-688, demonstrated that glycine is cleaved and donates the amino moiety to the penultimate amino acid, resulting in the amidation of the latter. The requirement for glycine as the amino group donor has been substantiated by other authors.
Landymore, A. E. N., et al, BBRC Vol. 117, #1, 1983, p. 289-293 demonstrated that D-alanine could also serve as an amino donor in the amidation reaction. Subsequent work by Kizer et al, PNAS, Vol. 81, 1984, p. 3228-3232, showed two distinct enzyme activities in rat brain which were capable of catalyzing the .alpha.-amidating reaction. The higher molecular mass species (70,000 daltons) has a specificity restricted for glycine at the carboxyl-terminus of the substrate. The lower molecular mass enzyme accepts a substrate with .beta.-alanine as the carboxyl-terminal amino acid.
The pH optimum for the .alpha.-amidating enzyme extracted and partially purified from porcine pituitary was reported by Bradbury A. F., and Smythe D. G., BBRC, Vol. 112, #2, 1983, p. 372-377 to be approximately 7.0. Eipper, B. A., et al, PNAS, Vol. 80, 1983, p. 5144-5148, corroborated these results by reporting a pH optimum of 7 for an .alpha.-amidating enzyme which was partially purified from rat pituitaries. They also noted that enzyme activity declined rapidly at pH levels below 6.5 or above 7.5.
In all of the aforementiond publications, which are incorporated herein by reference the extracts and partially purified enzyme mixtures contained additional proteolytic enzymes which degrade the potential substrate and products as well as the .alpha.-amidating enzyme.
It is therefore the object of the invention to provide a purified .alpha.-amidating enzyme which can efficiently be used to produce .alpha.-amidated peptides from peptide or polypeptide substrates, to prepare monoclonal antibodies specific for the enzyme, and to construct prokaryotes or other appropriate unicellular organisms or host cells isolated from multicellular organisms containing heterologous DNA coding for the enzyme. This and other objects of the invention will become apparent to those skilled in this art from the following detailed disclosure.